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Abstract:

A method for mixing and dispensing homogeneous compounds on a surface, at
least one reactant in a carrier fluid in laminar flow, and cell (3) for
implementing the method. The cell includes reaction chamber (16) having
at least one reaction surface (13) where the reactant can be fixed,
directly or indirectly, optionally reversibly, at least three fluid
inlets/outlets (25, 26), at least one fixed volume reservoir in
communication with the reaction chamber and outside the reaction chamber
via injection orifice (27), a fluid loop including at least one feeding
port, at least one extraction port and at least one reservoir whose
volume is variable and adapted to communicate independently with the
reaction chamber via each of its inlets/outlets; device for circulating
fluids in the reaction chamber and the fluid loop. The method and cell
can be used to prepare homogeneous surface films, or to carry out
"target-probe" identification reactions.

Claims:

1. A method for the homogeneous mixing and distribution, on a surface, of
at least one reactant carried by a carrier fluid in laminar flow,
comprising the following essential steps:a) a reaction chamber is
provided, which chamber has:at least one reaction surface on which the
reactant is capable of being fixed, directly or indirectly, optionally
reversibly,at least three fluid inlets/outlets, andat least one
reservoir, the volume of which is fixed, the fixed-volume reservoir being
able to communicate, firstly, with the reaction chamber and, secondly,
with the outside of the reaction chamber via an injection orifice;b)
optionally, the injection orifice of the fixed-volume reservoirs is
hermetically closed or kept hermetically closed and then at least one
fluid other than the carrier fluid containing the reactant is introduced
into the reaction chamber via at least one inlet/outlet of the reaction
chamber;c) the carrier fluid containing the reactant is injected into at
least one of the fixed-volume reservoirs;d) the carrier fluid containing
the reactant is circulated between the fixed-volume reservoir, the
reaction chamber and a variable-volume reservoir able to communicate
independently with the reaction chamber via each of the inlets/outlets of
the reaction chamber;e) step d) is repeated by successively selecting the
various inlets/outlets;f) optionally, steps b) and/or c) and/or d) and/or
e) are repeated.

2. The method as claimed in the preceding claim, in which steps d) and e)
respectively comprise:a substep d1) and e1) during which the fluid is
circulated from a fixed-volume reservoir to the variable-volume reservoir
via a first inlet/outlet of the reaction chamber, anda substep d2) and
e2) during which the fluid is circulated from a variable-volume reservoir
to a fixed-volume reservoir via a second inlet/outlet of the reaction
chamber that is different than the first inlet/outlet.

3. The method as claimed in claim 1, in which step b) comprises an
initiating step during which an initiating fluid is introduced into the
reaction chamber via at least one inlet/outlet of the reaction chamber.

4. The method as claimed in claim 1, comprising a final step during which
the reaction chamber and/or the variable-volume reservoir and/or the
fixed-volume reservoir(s) is (are) drained.

5. The method as claimed in the preceding claim, in which the final step
also comprises a step of releasing the reactants, and/or a step of
decontaminating the reaction chamber and/or the variable-volume reservoir
and/or the fixed-volume reservoir(s).

6. The method as claimed in claim 1, comprising at least one step of
flushing the gas bubbles present, preferably via the injection orifice of
a fixed-volume reservoir.

7. The method as claimed in claim 1, in which the reaction surface carries
a plurality of probes, in particular chemical or biochemical probes,
grafted onto said surface, and in which said reactants are capable of
reacting specifically with said probes.

8. The method as claimed in claim 1, in which the reactants are able to
form a homogeneous, in particular organic and/or inorganic chemical,
polymeric or electrochemical, film on the reaction surface.

9. A method of analysis, comprising the implementation of a method for
homogeneous mixing and distribution on a surface as claimed in claim 1,
further comprising a step of detecting the reactants fixed to the
reaction surface.

10. A cell for homogeneously mixing and distributing, on a surface, at
least one reactant carried by a carrier fluid in laminar flow,
comprising:a reaction chamber which has:at least one reaction surface on
which the reactant is capable of being fixed, directly or indirectly,
optionally reversibly,at least three fluid inlets/outlets, andat least
one reservoir, the volume of which is fixed, the fixed-volume reservoir
being able to communicate, firstly, with the reaction chamber and,
secondly, with the outside of the reaction chamber via an injection
orifice equipped with hermetic closure means,a fluid loop comprising at
least one feed port, at least one extraction port and at least one
reservoir, the volume of which is variable, the variable-volume reservoir
being able to communicate independently with the reaction chamber via
each of the inlets/outlets;means for circulating the fluids in the
reaction chamber and in the fluid loop.

11. The cell as claimed in the preceding claim, in which the volume of the
fixed-volume reservoir(s) is greater than or equal to the maximum volume
of the variable-volume reservoir.

12. The cell as claimed in claim 10, in which the inlets/outlets of the
reaction chamber are arranged regularly on the periphery of the reaction
chamber.

13. The cell as claimed in claim 10, in which the reaction chamber is
delimited at the top by a cover provided with said inlets/outlets, at the
bottom by said reaction surface and laterally by a leaktight seal.

14. The cell as claimed in the preceding claim, in which the lower face of
the cover comprises a peripheral groove in which a leaktight O-ring seal
is housed.

15. The cell as claimed in claim 13, in which the reaction surface is the
upper surface of a fitted part, preferably a microscope slide, the cell
also comprising means for positioning the fitted part relative to the
cover.

16. The cell as claimed in claim 10, further comprising means for
regulating the temperature in the chamber and/or means for regulating the
temperature in the fluid loop.

17. The cell as claimed in the preceding claim, in which the means for
regulating the temperature are in the form of at least one Peltier-effect
cell.

18. The cell as claimed in claim 13, comprising means for locking the
cover relative to the reaction surface.

19. The cell as claimed in claim 10, also comprising means for detecting
the reactants fixed to the reaction surface.

20. A chemical and/or biochemical "target-probe" recognition apparatus,
comprising at least one cell as claimed in claim 10, in which the
reaction surface of the cell is in the form of a microarray of specific
probes, prepared on a support, preferably a microscope slide, and in
which the carrier fluid contains a plurality of "target" reactants
capable of reacting specifically with the probes of the microarray.

21. An apparatus for forming a homogeneous film on a surface, comprising
at least one cell as claimed in claim 10.

22. The apparatus as claimed in claim 20, comprising:a device for
distributing at least one fluid in the fluid loop of the mixing and
distributing cell;means for injecting at least one carrier fluid
containing said reactant into the reaction chamber of the mixing and
distributing cell.

23. The apparatus as claimed in claim 20, in which a plurality of mixing
and distributing cells are placed in parallel and/or in series.

24. An analytical apparatus comprising an apparatus as claimed in claim 20
and means for detecting the reactants fixed to the reaction surface, the
detection means being possibly attached to the mixing and distributing
cell(s).

Description:

FIELD OF THE INVENTION

[0001]The invention relates to the field of automatic apparatuses for the
optimization of reactions on surfaces. In particular, the invention
relates to a method for the homogeneous mixing and distribution, on a
surface, of at least one reactant carried by a carrier fluid in laminar
flow, and also to a cell for implementing this method.

[0002]The invention also relates to an automatic apparatus for carrying
out biological, biochemical and chemical reactions in a homogeneous and
reproducible manner on a planar or porous surface.

CONTEXT OF THE INVENTION

[0003]The fields of genomics and of post-genomics are currently in full
expansion. This is closely linked to the development of new tools for
high-throughput analysis. Biochips (DNA, protein, aptamers, etc.) are
part of these powerful analytical tools. Biochips comprise a support on
which biological or chemical molecules are localized and immobilized
(from a few tens to several thousand per cm2). These molecules,
usually called "probes", have the ability to specifically recognize
molecules in solution. Each probe has the ability to interact more or
less specifically and more or less strongly with another biological or
chemical molecule called "target".

[0005]These techniques make it possible to screen a large amount of
different molecules simultaneously. They are essential for the generation
of new sources of information for biologists. In fact, the biological
information making it possible to decipher molecular motifs associated
with a pathology, to determine the level of gene expression in response
to a stress (in particular toxicology) or else to search for
polymorphisms related to genetic diseases will only be obtained using
these such multiple, parallel and simultaneous screening platforms.

[0006]Another factor to be taken into account is that of the sensitivity
of the tests. In fact, biological products (for example, nucleic acids,
proteins, organic molecules, etc.) are extracted in minute amounts. In
order to optimize the sensitivity and duration of analysis, it is sought
to miniaturize the analytical devices. For example, DNA chips are known
in which several hundred thousand probes are immobilized on 1 cm2. A
concept in full expansion in the micro- and nanotechnology field is the
lab-on-chip.

[0007]A great deal of effort has thus been given over the past few years
to the fabrication of these devices. However, few tests are routinely
used in biological laboratories. The "young" biochip technology, for
example, suffers from a lack of reliability and reproducibility. At the
current time, only one example of a chip has received CE-IVD
certification (AmpliChip® CYP450, Roche). The use of chips for
molecular and genetic diagnosis in a clinical laboratory, firstly, means
that the results must be standardized and made reliable and, secondly, it
must allow high-throughput analyses.

[0008]For this, all the steps, from the fabrication of the support to the
reading of the chips and to the data processing, must be made reliable.
There is an abundant literature relating to the control of processes for
developing microarrays, various methods of obtaining target strands in
the case of nucleic acid hybridization, and also data processing.
However, few studies relate to the impact of the hybridization step on
the biological results.

[0009]Two parameters appear to be important: the uniformity of the
hybridization over the entire support and the automation of the process.
To achieve excellent homogeneity, it is essential to give each target in
solution the same probability of "seeing" all the probes attached to the
support. The massive amounts of analyses required in the context of
genetic diseases (for screening, for example) mean that it is necessary
to have automatic tools. This aspect is important since it makes it
possible to improve and guarantee good repeatability and reproducibility
of the experiments, thereby reducing the multiple sources of variations.

[0010]Chips appear to be powerful analytical tools since several hundred
reactions can be carried out in parallel on the same support. However, in
order for all these analyses to be comparable, it is necessary for them
to be subjected to the same reaction, on the scale of the probe.

[0011]In the laboratory, hybridizations on a glass slide are commonly
carried out between slide and cover slip ("passive hybridization"
method). The hybridization solution is therefore fixed (static) and this
type of procedure is accompanied by a certain number of imperfections. In
particular, this conventional hybridization method is limited by the
diffusion of the targets in the hybridization buffer at the surface of
the support on which the probes are immobilized (brownien movement only).

[0012]It is found that the fact of introducing a mixture into the fluids
can have a positive effect on the hybridization results. Several
principles of micro-mixing associated with DNA chips have been studied.
The idea is to be free of the molecular diffusion by causing a mass
transfer of the DNA molecules.

[0013]A first approach is based on the migration of the charged DNA
molecules under the action of an electric field (Edman et al. (1997)
"Electric field directed nucleic acid hybridization on microchips"
Nucleic Acid Res. 25(24): 4907-14). This method has given results 30 to
40 times superior to a passive hybridization method.

[0014]Other approaches for accelerating the hybridization make use of mass
transfers by convection. A first approach is based on the generation of
convection cells via acoustic waves (Liu et al. (2003) "Hybridization
enhancement using cavitation microstreaming" Anal. Chem. 75(8): 1911-7).
The micromixing is generated directly in the solution at the surface of
the slide without the introduction of dead volumes. It has been
demonstrated that this method improves the intensity of the hybridization
signal and the kinetics by a factor of 5 compared with a static
hybridization. The authors indicate a gain in homogeneity without giving
results in terms of numbers.

[0015]The alternative consists in creating a mechanical agitation. These
microfluidic systems (McQuain et al. (2004) "Chaotic mixer improves
microarray hybridization" Anal Biochem. 325(2): 215-26) prove to be
difficult to implement despite the simplicity of the principle. Compared
with a static method, this method increases by 2- to 8-fold the
hybridization effectiveness according to the probe density, the target
concentration and the hybridization buffer volume. McQuain et al. (see
also WO-A-03/16547) have developed a glass slide agitation system based
on the principle of chaotic advection. They state that there is an
improvement in the uniformity of the signal close to the uniformity of
the immobilization of the probes on the support (variation coefficient of
19%) and a factor of 2 compared with a static mode.

[0016]Some of these techniques require specific supports (nanogen
platform) or else expensive transducers. In addition, these systems are
not integrated into automated systems, which does not make it possible to
guarantee the use of small reaction volumes (comparable to systems
between slide and cover slip 40-50 μl). These techniques deal more
with the problem of the hybridization according to a kinetic aspect and
for the most part neglect the aspects of automation and uniformity of the
hybridization on the scale of the chip.

[0017]Conversely, other teams work on the access of DNA chips for mass
analysis. The techniques currently used in this type of test are well
plates. However, each well consumes a considerable volume of biological
material and homogeneity is not ensured due to the absence of controlled
agitation within the well.

[0018]A certain number of commercially available hybridization stations
exist which make it possible to carry out automatic hybridizations. The
volumes involved are very large (greater than 200 μl). Most of these
stations perform liquid drain and fill movements with regard to the
surface, which result in a laminar flow according to preferential
pathways. This is accompanied by a nonuniformity of the reaction zones.
McQuain et al. demonstrated the importance of creating homogeneous
agitation. It is therefore important to control the manner in which the
mixing is carried out.

[0019]It has been proposed to use standardized and relatively inexpensive
microscope slides and to carry out mixing of the fluids by chaotic
advection. The flows of liquid inside a microfluidic reaction chamber
(chamber thickness less than 100 μm) follow laminar flows (very low
Reynolds number). As a result, poor mixing takes place within a
microchamber: the first principle consists in introducing a temporal flow
variation. This is carried out by injecting the fluid at various sites
periodically over time. Furthermore, in this type of periodic
two-dimensional flow over time, certain regions of the fluid may resist
the appearance of chaos. The introduction of a three-dimensionality of
the flow, at the level of the injections, then makes it possible to
eliminate these dead zones and to promote mixing over the entire surface
of the slide. Simulations show that, by means of this method of mixing by
chaotic advection, the target diffusion layer is reduced, which makes it
possible to miniaturize the device and to apply it to diagnostic chips
(biochips), in the context of a high-throughput detection.

OBJECTIVES OF THE INVENTION

[0020]The objective of the invention is to overcome the deficiencies of
the devices of the prior art. In particular, it is a question of making
the hybridization technique reliable, and more generally of making
reliable the homogeneous mixing and distribution, on a surface, of at
least one reactant carried by a carrier fluid. An objective of the
invention is to develop an automatic high-throughput tool for this
purpose.

[0021]Another objective is to propose a method for the homogeneous mixing
and distribution, on a surface, of at least one reactant carried by a
carrier fluid, which uses the phenomenon of chaotic advection generated
in a laminar flow.

[0022]Another objective of the invention is to propose an automatic tool
for depositing a chemical, biochemical or alternatively polymer coating,
on a planar or porous surface, the thickness and homogeneity of which
coating are reproducibly controlled.

[0023]An objective of the invention is to develop a method for the purpose
of improving the homogeneity and the reproducibility of the chemical,
biochemical and biological reaction over the whole of a reaction surface.

[0024]Another objective is to develop a cell for the homogeneous mixing
and distribution, on a surface, of at least one reactant carried by a
carrier fluid, in which the variability of the reaction between two
points of the reaction surface is reduced.

[0025]An additional objective of the invention is to provide a method and
a device that can be used in particular in the case of
diagnosis--"biochips"--the fabrication and the use of which are
relatively inexpensive.

[0026]Yet another objective of the invention is to propose a method that
is reversible, in the sense that it may be possible to separate the
reactants from the reaction surface.

[0027]The specifications of an automatic tool imply: [0028]working with
as small a volume of liquid as possible: for a unit surface, the volume
of liquid is determined by the thickness of liquid with regard to the
surface; [0029]agitating the reactants in solution in order to improve
the distribution of these reactants with respect to the surface, so as to
obtain a homogeneous concentration on the surface; [0030]creating a
homogeneous circulation of the liquid on the surface, so as to prevent
the appearance of "dead" zones; [0031]controlling the reaction
temperature; [0032]automatically conveying several liquids;
[0033]automatically emptying the device; [0034]being able to directly
inject reactants of any type into the reaction device at any moment.

[0035]Another objective is to propose an analytical apparatus for
detecting the state of advancement of the reaction, in order to optimize
the duration of the reaction, both in the case of a target-probe
recognition reaction and in the case of the formation of a coating, on a
reaction surface. [0036]An additional objective of the invention is to
propose an apparatus for analyzing a sample, for example a biological
sample. Another objective is to provide an apparatus for controlling the
quality of a coating on a reaction surface.

BRIEF DESCRIPTION OF THE INVENTION

[0037]It is to the inventors' credit to have developed, firstly, a method
and, secondly, a cell for homogeneously mixing and distributing, on a
surface, at least one reactant carried by a carrier fluid. In particular,
such a cell may be integrated into various apparatuses, in particular a
chemical and/or biochemical recognition apparatus of the probe-target
type, or an apparatus for forming a homogeneous film on a surface,
themselves capable of comprising means for detecting the advancement of
the reactions.

[0038]In order to set up this technology, the inventors have produced a
fluidic system equipped with an automated hybridization chamber which
makes it possible to carry out a method based on the principle of chaotic
advection within the chamber.

[0039]Thus, the invention, as defined in the claims, relates firstly to a
method for the homogeneous mixing and distribution, on a surface, of at
least one reactant carried by a carrier fluid in laminar flow, comprising
the following essential steps: [0040]a) a reaction chamber is provided,
which chamber has: [0041]at least one reaction surface on which the
reactant is capable of being fixed, directly or indirectly, optionally
reversibly, [0042]at least three fluid inlets/outlets, and * at least one
reservoir, the volume of which is fixed, the fixed-volume reservoir being
able to communicate, firstly, with the reaction chamber and, secondly,
with the outside of the reaction chamber via an injection orifice;
[0043]b) optionally, the injection orifice of the fixed-volume reservoirs
is hermetically closed or kept hermetically closed and then at least one
fluid other than the carrier fluid containing the reactant is introduced
into the reaction chamber via at least one inlet/outlet of the reaction
chamber; [0044]c) the carrier fluid containing the reactant is injected
into at least one of the fixed-volume reservoirs; [0045]d) the carrier
fluid containing the reactant is circulated between the fixed-volume
reservoir, the reaction chamber and a variable-volume reservoir able to
communicate independently with the reaction chamber via each of the
inlets/outlets of the reaction chamber; [0046]e) step d) is repeated by
successively selecting the various inlets/outlets; [0047]f) optionally,
steps b) and/or c) and/or d) and/or e) are repeated.

[0048]Advantageously, for the implementation of this method, the inventors
have developed a cell for homogeneously mixing and distributing, on a
surface, at least one reactant carried by a carrier fluid in laminar
flow, comprising: [0049]a reaction chamber which has: [0050]at least
one reaction surface on which the reactant is capable of being fixed,
directly or indirectly, optionally reversibly, [0051]at least three fluid
inlets/outlets, and [0052]at least one reservoir, the volume of which is
fixed, the fixed-volume reservoir being able to communicate, firstly,
with the reaction chamber and, secondly, with the outside of the reaction
chamber via an injection orifice equipped with hermetic closure means,
[0053]a fluid loop comprising at least one feed port, at least one
extraction port and at least one reservoir, the volume of which is
variable, the variable-volume reservoir being able to communicate
independently with the reaction chamber via each of the inlets/outlets;
[0054]means for circulating the fluids in the reaction chamber and in the
fluid loop.

[0055]The method and the cell according to the invention satisfy the major
requirements of the specifications mentioned above. In fact, they make it
possible to develop mixing by chaotic advection within the film of
carrier liquid in contact with the reaction surface. Thus, the carrier
liquid, and therefore the reactants that it carries, are distributed
homogeneously over the entire reaction surface. In other words, all the
points of the reaction surface are equivalent: no preferential flows of
carrier fluid exist on the reaction surface.

[0056]For example, in the case of a probe-target recognition reaction (for
example, by hybridization), each target has the same probability of
seeing the probes immobilized on the reaction surface. In the case of the
formation of a coating, the reactants intended to form the coating are
distributed homogeneously over the entire reaction surface to be coated.

[0068]In FIGS. 9, 10 and 11, the arrows indicate the direction of
circulation of the fluids and the symbol A indicates that the
corresponding valve is blocking.

[0069]FIG. 12 is a graph representing the signal/noise ratio (SNR) for two
types of probes exhibiting a single nucleotide polymorphism, and in two
hybridization techniques (technique between slide and cover slip,
compared with the dynamic technique of chaotic mixing according to the
invention), as explained in the examples (signal/noise ratio; shown as
black: allele a; shown as hatched: allele b).

[0070]FIG. 13 illustrates the overall kinetics of hybridization for the
static and dynamic hybridizations, as explained in the examples (average
fluorescence [arbitrary units, a.u.] as a function of time [min]; black
square: static hybridization; circle: dynamic hybridization according to
the invention).

DETAILED DESCRIPTION OF THE INVENTION

[0071]The invention will now be described with reference to the figures
mentioned above, essentially in the case of an automatic apparatus for
hybridization on a DNA chip. Of course, other types of molecular
recognition may be contemplated, for example chosen from the following
probe/target pairs: DNA/DNA, DNA/RNA, RNA/RNA, PNA/DNA, PNA/RNA,
PNA/protein, protein/DNA, protein/RNA, protein/protein, chemical molecule
(for example, hormones, lipids, glycolipids, carbohydrate)/protein,
chemical molecule/DNA, etc. (PNA: peptide nucleic acid).

[0072]Of course, this list is in no way limiting, given that the problem
that the invention is intended to solve is that of the homogeneous
distribution and mixing, on a reaction surface, of at least one reactant
carried by a carrier fluid. In this case, reference will be made to
indirect fixing of the reactant--the target--to the reaction surface, via
probes immobilized on said surface.

[0073]The invention can also be applied to the deposition of reactants in
order to form a homogeneous chemical, biochemical or in particular
polymer film, on a reaction surface, the surface condition of which may
have been modified beforehand in order to improve the deposition of the
reactants. In this case, the reactant can be directly or indirectly fixed
to the reaction surface, as appropriate.

[0074]The term "carrier fluid" denotes a fluid containing the reactants,
i.e., in particular, the fluid which is injected into the reaction
chamber via the injection orifice. Moreover, the reaction chamber
generally contains an initiating fluid, for example, before the injection
of the carrier fluid. As soon as the initiating fluid and the injected
carrier fluid mix, the mixture is generally denoted as carrier fluid.

[0075]FIG. 1 shows an isometric perspective view of an automatic
hybridization apparatus 1 for hybridization on DNA chips in the
microscope slide format. This apparatus comprises a cover 2, for example
made of plastic, and a hybridization cell 3 into which it is possible to
insert any type of microscope slide (format 1 inch×3 inch×1
mm, i.e. 26 mm×76 mm×1 mm) on which biological probes have
been immobilized. The cell confines the surface to be hybridized
(reaction surface on which the probes are immobilized) in a reaction
chamber of small volume in which the processes relating to the
hybridization will take place. This chamber is connected via the tubes 4
to a fluid loop 6 illustrated in FIG. 2. The fluid loop comprises
microelectro-fluidic elements preferably located under the cover 2. These
elements make it possible to carry out mixing by chaotic advection in the
reaction chamber.

[0076]Advantageously, in order to control the reaction conditions,
temperature regulating means make it possible to regulate the temperature
of the cell, in the reaction chamber and/or in the fluid loop. This
involves, for example, one or more heating elements or one or more
Peltier-effect cells.

[0077]FIG. 2 also shows a distribution system 8 which makes it possible to
distribute various solutions in the fluid loop 6 in order to carry out,
for example, prehybridizations, hybridizations and/or washes. The
solutions are conveyed to the distribution system 8 via the tubes 5.

[0078]The electrofluidic elements of the fluid loop and the distribution
system are preferably controlled by power boards 9 protected by the
cover. The temperature regulating means and also the power boards 9 are
supplied by a power supply 7 under the cover which converts the
alternating current of the mains supply to a 24V direct current (where
appropriate).

[0079]Advantageously, the power boards and the temperature regulating
means are controlled by an electronic board which has digital and analog
inputs and outputs. This board is mounted on a PCI port for computers.
Software controls the board which sends the information to the apparatus
1 via a cable connected to a communication port. Of course, other types
of communication ports can be contemplated.

[0080]As indicated above, the invention relates to a cell 3 for
homogeneously mixing and distributing, on a surface 13, at least one
reactant carried by a carrier fluid in laminar flow, comprising:
[0081]a reaction chamber 16 which has: [0082]at least one reaction
surface 13 on which the reactant is capable of being fixed, directly or
indirectly, optionally reversibly, [0083]at least three fluid
inlets/outlets 25, 26, and [0084]at least one reservoir, the volume of
which is fixed, the fixed-volume reservoir RF being able to communicate,
firstly, with the reaction chamber 16 and, secondly, with the outside of
the reaction chamber via an injection orifice 27 provided with hermetic
closure means 28, [0085]a fluid loop 7 comprising at least one feed
port 63, at least one extraction port 62 and at least one reservoir RV,
the volume of which is variable, the variable-volume reservoir RV being
able to communicate independently with the reaction chamber via each of
the inlets/outlets 25, 26; [0086]means for circulating the fluids in the
reaction chamber and in the fluid loop.

[0087]Advantageously, as will be specified during the description of the
method according to the invention, the volume of the fixed-volume
reservoir(s) RF is greater than or equal to the maximum volume of the
variable-volume reservoir RV.

[0088]Preferably, the inlets/outlets 25, 26 of the reaction chamber 16 are
arranged regularly on the periphery of the reaction chamber. This makes
it possible in particular to improve the homogeneity of the mixing and of
the distribution of the carrier fluid along the reaction surface.

[0089]According to a preferred embodiment illustrated in FIGS. 3 to 6, the
reaction chamber 16 is delimited at the top by a cover C provided with
said inlets/outlets 25, 26, at the bottom by said reaction surface 13 and
laterally by a leaktight seal 22. Advantageously, the lower face of the
cover C comprises a peripheral groove 21 in which a leaktight O-ring seal
22 is housed.

[0090]Preferably, the reaction surface is the upper surface of a fitted
part, for example a microscope slide, the cell also comprising means for
positioning the fitted part relative to the cover C.

[0091]FIG. 4 shows a front view of a cover C which constitutes the upper
part of the reaction chamber 16. The cover C comprises a rectangular
groove 21 in which an EPDM O-ring seal 22 is housed. The groove 21
delimits a rectangular inner surface 23 (in this case, 50 μm deep)
which represents the top of the reaction chamber 16. When the cover C is
applied to the microscope slide 13 as shown in FIG. 6, the outer surface
24 of the cover, delimited by the groove 21, is pushed against the
microscope slide 13. Thus, irrespective of the thickness of the slide 13,
the height (h) of the chamber 16 does not vary. When the surface 24 is
pushed against the slide 13, the O-ring seal is squashed into the groove
21. This makes it possible to ensure the leaktightness of the chamber 16.
In this example, the chamber comprises four inlets/outlets 25, 26 located
at the four corners of the chamber. These inlets/outlets make it possible
to circulate the carrier fluid and the reactants between the fixed-volume
reservoir RF and the variable-volume reservoir RV, along the reaction
surface. In addition, these inlets/outlets are connected to at least one
feed port and at least one extraction port, which allow the injection and
the extraction of the reactant solutions, washing solutions and
decontaminating solutions in the device. An injection orifice 27 makes it
possible to inject the biological material into the reaction chamber,
during a step when the device is functioning, from the outside. This
injection orifice 27 communicates with a fixed-volume reservoir RF. A
stopper 28 (FIG. 6) makes it possible to hermetically close the
fixed-volume reservoir RF with respect to the outside, in particular for
the phases of emptying and filling the reaction chamber 16.

[0092]FIG. 3 shows a detailed isometric view of the cell 3 when it is
open. The cell comprises a microscope slide support 12, for example made
of fortal, on which is placed a microscope slide 13 provided with a
microarray of biological probes. The slide is positioned in the
horizontal plane by virtue of two abutment strips 14 and 15 machined on
the support 12. The cell comprises two levers 17 connected to the support
12 via a common shaft 18 which ensures a pivot connection between the
levers 17 and the support 12. The cover C is connected to the levers 17
via a shaft 18. This mechanism makes it possible to apply the cover C
reproducibly to the microscope slide with a uniformly distributed force.
Thus, this reduces the risks of the support breaking and this guarantees
excellent leaktightness. A locking system 19 at the end of the levers 17
makes it possible to lock the system reproducibly with a sufficient
force. A heating element 20 of the thermofoil type is bonded to the
support 12 and makes it possible to control the hybridization
temperature.

[0093]FIG. 6 shows a sectional view of the cell according to the
invention. The bore 29 in the support 12 accepts a probe which makes it
possible to measure the temperature of the reaction chamber 16 in
proximity to the reaction surface 13, in order to be able to regulate the
temperature as well as possible. FIG. 6 makes it possible to observe the
locking system 19. The shaft 31 slides in the part 30 via a polymer
bushing 34. The shaft 31 is kept in place on the part 30 by virtue of the
force of a spring 32 prestressed by the handle 33. To lock the cell, it
is necessary to exert a force on the handle 33 so as to slide the shaft
31 into the part 30 and it is then necessary to turn the handle 33
through 90° (for example) in order to turn the shaft 31 through
the same angle. The lug 35 pushes up against the part 36 which is
detached as one to the support 12. Preferably, this part 36 is
manufactured in a metal that is harder than the material of the support,
in order to limit wearing. The system is then locked and the force of the
spring 32 which keeps the system 19 locked is exerted on the cover C via
the levers 17 and the shaft 18. This force is sufficient to squash the
0-ring seal 22 so that the surface 24 of the cover 16 is pressed against
the microscope slide. The levers 17 make it possible to uncouple the
force to be exerted on the chamber in order to allow a user to supply
this force more comfortably.

[0094]FIG. 7 shows the scheme of the distribution system 8 and of the
fluid loop 7 connected to the reaction chamber 16. The solutions used for
the hybridization operations are contained in bottles 37. The solutions
are conveyed via the tubes 5 to a mixing valve 38 by virtue of a pump 39
placed downstream of the valve. It is preferably a membrane pump
connected to two nonreturn valves. It may also be a solenoid valve. The
mixing valve 38 makes it possible to select the solution to be used for
priming the system or filling the fluid loop. The outlet of the pump 39
is connected to the fluid loop via a four-way fluid connector 40. The
fluid loop comprises four valves 42, 43, 44, 45 connected respectively to
the inlets/outlets 57, 56, 54, 55 of the chamber 16. The valves 42 and 43
are connected via the four-way fluid connector 40 to the outlet of the
pump 39 and to the inlet of the pump 46. The valves 44 and 45 are
connected to an outlet valve 47 of the loop and to the outlet of the pump
46 via the four-way fluid connector 41. A waste container 48 is placed
downstream of the outlet valve 47 of the loop in order to recover the
solutions used via a tube 5'.

[0095]Advantageously, the pump 46 is in the form of a solenoid valve of
variable volume, which constitutes the variable-volume reservoir RV.

[0096]For the phases of filling the fluid loop including the chamber 16,
the solution is selected by virtue of the mixing valve 38, and the pump
39 conveys the solution to the loop inlet. The valves 42, 43, 44 and 45
are then closed and the pump 46 and the valve 47 are opened. The pump 39
then circulates the solution toward the waste container 48. A first part
of the fluid loop is filled. In order to fill the other part, the pump 46
must be closed and the valves 42, 43, 44, 45 and 47 must be opened, the
pump 39 then circulates the solution toward the waste container, causing
the solution to enter the chamber 16 via the inlets/outlets 57, 56 and
causing the solution to exit via the inlets/outlets 54, 55. In order to
fill the second part of the loop, the injection orifice of the chamber
must be closed because the solution is pushed by the pump 39. Similarly,
the fluid loop can be emptied by selecting air instead of a solution.

[0097]For the mixing phases, the pump 39 is closed, and the valve 47 is
also closed. On the other hand, the injection orifice 27 is open or
optionally closed. The mixing is carried out by a series of fluid
circulation phases. In a first half of the phase, the valve 43 is open
and the valves 44, 45 and 42 are closed. In the high state, the pump 46
draws up the solution via its inlet and extracts the solution from the
chamber 16 via the inlet/outlet associated with the valve 43, which is
open, while the other valves 44, 45 and 42 are closed. The chamber is
always full because the volume of solution extracted is compensated for
by the volume of solution contained in the fixed-volume reservoir RF
which communicates with the injection orifice 27.

[0098]Then, in the second half of the phase, the valve 44 is open and the
valves 42, 44 and 45 are closed. The pump goes from the high state to the
low state. In the low state, the pump pushes the part of solution drawn
up in the high state and injects the solution into the chamber 16 via the
inlet/outlet associated with the valve 44, which is open, while the other
valves 42, 44 and 45 are closed. A part of the volume of the chamber
equal to the volume displaced by the pump 46 is injected into the
fixed-volume reservoir, via the injection orifice.

[0099]The volume displaced by the pump 46 does not exceed the volume of
the injection orifice so that, during the high state, no air is injected
into the chamber and therefore into the loop. In fact, this can bring
about head losses and poor mixing. In addition, this prevents, during the
low state, the solution which fills the chamber from leaving the chamber
via the injection orifice.

[0100]The pump 46 will perform a series of pulses characterized by a high
state and a low state, during which the inlet/outlet connected to the
pump 46 during the passage from the high state to the low state of the
pump 46 and then from the low state to the high state, will change from
one phase to the next. Consequently, the variable-volume reservoir can
communicate with the reaction chamber independently via each of the
inlets/outlets.

[0101]During the mixing, it is possible to inject biological material into
the injection orifice 27 provided that there is no overspill in the low
state.

[0102]Furthermore, the injection orifice operates as a bubble trap. In
fact, the solutions commonly used for hybridization contain surfactants
which make it very difficult to inject them without bubbles. Given that
the injection orifice is released, the bubbles which enter therein are
not reinjected into the chamber.

[0103]According to an advantageous characteristic of the invention, the
cell is provided with means for detecting the reactants fixed to the
reaction surface. For example, in the case of a nucleic acid
hybridization, said means may be a fluorescence scanner.

[0104]More generally, it is possible to contemplate any type of sensor
capable of detecting the presence of reactants at a given position on the
reaction surface. The sensor is connected to an image analyzing device,
for example, in order to determine the positions on the reaction surface
where a reactant has effectively been fixed. In the case of microarrays,
where a probe-target recognition is involved, the characteristics of the
probes are known, which makes it possible to deduce the characteristics
of the targets--the reactants fixed--and therefore, ultimately, those of
the sample analyzed.

[0105]Moreover, according to the experimental conditions and the type of
sensor, the analysis may be qualitative (presence or absence of a
target), quantitative (amount of target present, correlated for example
to a coloration or fluorescence intensity), or semi-quantitative
(quantitative above a certain level of detection, and qualitative below
this level of detection).

[0106]It is thus possible to have a dynamic analysis tool. For example,
the hybridization reactions, or more generally the probe-target
recognition reactions, are carried out by repeating phases of mixing and
distributing the carrier fluid on the reaction surface, and the fixing of
targets to the reaction surface is simultaneously detected using an
appropriate sensor. Then, when it is determined that the situation
between two detections/analyses has no longer changed for a predetermined
number of cycles, i.e. no new recognition reaction has been detected, it
may be considered that the analysis is complete. This makes it possible
to reduce the duration of the analyses, since the mixing cycle number is
not arbitrarily fixed, but depends on the situation effectively measured.

[0107]The analysis may also relate to the homogeneity of a coating. It is
possible, for example, to measure the coloration of the reaction surface
after having deposited a chemical, biochemical and/or polymeric film. It
is also possible to contemplate measuring the variations in the intensity
of a light beam passing through the film and the support. If the
variability of the intensity of this light beam is less than a
predetermined threshold, it may be considered, for example, that the film
is homogeneous in terms of thickness. A prior calibration will make it
possible to determine the thickness of this film.

[0108]Thus, a tool is provided which makes it possible to control the
quality of a coating on a support such as a microscope slide.

[0109]Consequently, the invention also relates to an apparatus for
carrying out chemical and/or biochemical "target-probe" recognition
reactions, comprising at least one cell in accordance with the invention,
in which the reaction surface of the cell is in the form of a microarray
of specific probes, prepared on a support, preferably a microscope slide.
In this case, the carrier fluid contains a plurality of "target"
reactants capable of reacting specifically with the probes of the
microarray.

[0110]The invention also relates to an apparatus for forming a homogeneous
film on a surface, comprising at least one cell in accordance with the
invention.

[0111]Preferably, these apparatuses comprise a device for distributing at
least one fluid in the fluid loop of the mixing and distributing cell. A
distributing device, for example a manifold, makes it possible to
automatically distribute the fluid(s), buffer solution, cleaning
solution, rinsing solution and the like, and air, used during the
operating of the apparatus. According to another characteristic of the
invention, means for injecting at least one carrier fluid containing said
reactant into the reaction chamber of the mixing and distributing cell,
via the injection orifice of the fixed-volume reservoir, are provided.
Here again there is a possibility of automation, which improves the
reproducibility.

[0112]It is entirely possible to contemplate having a plurality of mixing
and distributing cells in parallel and/or in series. This may be useful
for carrying out several recognition reactions, or several film
depositions, in parallel, or even successively on the same reaction
surface.

[0113]In addition, the invention relates to an analytical apparatus
comprising at least one cell in accordance with the invention and means
for detecting the reactants fixed to the reaction surface. The detection
means may be attached to the analytical cell, or attached to the
apparatus. Thus, various types of mixing and distributing cells may be
contemplated, each comprising a reaction surface and detection means
suitable for certain reactants, and all compatible with the same
apparatus. This may prove to be useful when the same user must be able to
carry out a large variety of reactions and analyses.

[0114]On the other hand, when the reaction studied is always the same, it
may be preferable to attach the detection means to the analytical
apparatus rather than the mixing and distributing cell, in particular to
make the analyses less expensive and more reproducible.

[0115]The invention also relates, as was described above in relation to
the operating of the device according to the invention, to a method for
the homogeneous mixing and distribution, on a surface, of at least one
reactant carried by a carrier fluid in laminar flow. This method
comprises essentially the following steps.

[0116]First of all, a reaction chamber as described above is provided.
Optionally, at least one fluid other than the carrier fluid containing
the reactant is introduced into the reaction chamber via at least one
inlet/outlet of the reaction chamber, for example in order to initiate
the method, or to fill the fluid loop.

[0117]Next, the carrier fluid containing the reactant is injected into at
least one of the fixed-volume reservoirs. Thus, the carrier fluid
containing the reactant will be able to circulate toward the reaction
chamber, via the injection orifice. The injection orifice of the
fixed-volume reservoir is then closed or left open, and then the carrier
fluid containing the reactant is circulated between the fixed-volume
reservoir, the reaction chamber and a variable-volume reservoir able to
communicate independently with the reaction chamber via each of the
inlets/outlets of the reaction chamber. This operation can be carried out
by placing the variable-volume reservoir in an empty state beforehand,
and then filling it from the reaction chamber via an inlet/outlet. A
suction which will empty the fixed-volume reservoir is thus created. This
step is then reproduced, in the opposite direction, by circulating the
fluid via another inlet/outlet, which makes it possible to distribute it
toward another zone of the reaction surface.

[0118]Thus, preferably, it is possible to distinguish two substeps
(without the order in which they are stated being obligatory): [0119]a
substep during which the fluid is circulated from a fixed-volume
reservoir to the variable-volume reservoir via a first inlet/outlet of
the reaction chamber, and [0120]a substep during which the fluid is
circulated from a variable-volume reservoir to a fixed-volume reservoir
via a second inlet/outlet of the reaction chamber that is different than
the first inlet/outlet.

[0121]In other words, for each step, a pair of inlets/outlets is selected.
In the first substep, the fluid circulates in one direction through one
of the two inlets/outlets of the pair, and then in the second substep,
the fluid circulates in the opposite direction through the other of the
two inlets/outlets of the pair. These two substeps are then repeated,
choosing another pair of inlets/outlets, i.e. a pair which differs from
the preceding pair by one or two inlets/outlets.

[0122]The previous steps are repeated, with the various inlets/outlets
being successively selected. Optionally, other samples of carrier fluid
containing the reactant (or a reactant other than that/those previously
used) can be injected via the injection orifice of the fixed-volume
reservoir, and the steps of circulating the fluids between the
fixed-volume reservoir and the variable-volume reservoir can be repeated.

[0123]In certain cases, it may be necessary to flush the gas bubbles
present from the device, preferably via the injection orifice of a
fixed-volume reservoir. This may be carried out by leaving the injection
orifice of a fixed-volume reservoir open and carrying out the mixing,
such that the gas bubbles are entrained to the fixed-volume reservoir and
then evacuated. In fact, the fixed-volume reservoir constitutes a high
point in the reaction chamber.

[0124]Finally, the method may comprise a final step during which the
reaction chamber and/or the variable-volume reservoir and/or the
fixed-volume reservoir(s) is (are) drained.

[0125]Optionally, when the reaction between the reactants and the reaction
surface is reversible, the final step may comprise a step for releasing
the reactants. It may also be desirable to decontaminate the reaction
chamber and/or the variable-volume reservoir and/or the fixed-volume
reservoir(s). These operations are carried out by circulating the
appropriate fluids in the fluid loop and in the reaction chamber, in a
manner similar to that used for the priming.

[0126]The method will be described in detail, with reference to the
schematic FIGS. 8 to 11. FIG. 8A represents a planar reaction surface 50
which comprises a micro-array 51 where biological probes have been
immobilized.

[0127]A cover 53 confines the surface 51 in a small volume, which
constitutes the reaction chamber. The chamber comprises at least four
ports, including: three inlets/outlets and one injection orifice. In the
case illustrated in FIG. 8, four inlets/outlets 54, 55, 56, 57 are
distributed regularly close to the periphery of the reaction chamber and
an injection orifice 65 is located close to the barycenter of the
inlets/outlets. Each inlet/outlet 54, 55, 56 or 57 is connected to a
fluid conduit 58, 59, 60 or 61 comprising an on/off valve, preferably a
solenoid valve. The conduits 58, 59, 60 and 61 are connected to a
variable-volume reservoir 64 which operates as a blocked valve when its
volume is at a minimum. The conduits, the chamber and the variable volume
constitute a fluid loop which also comprises a feed port 63 directly
connected to the conduits 60 and 61 and an extraction port 62 directly
connected to the conduits 58 and 59. The feed port 63 and the extraction
port 62 each comprise an on/off valve. The injection orifice 65 of the
chamber is connected to a fixed-volume reservoir 66, the volume of which
is greater than or equal to the maximum volume of the variable-volume
reservoir 64. The fixed-volume reservoir is provided with a leaktight
closure system.

[0128]The hybridization method consists in: [0129]1/ filling the fluid
loop with a buffer solution and bringing to temperature; [0130]2/
injecting the biological targets; [0131]3/ extracting from the fluid loop
a part of the buffer solution having the same volume as the volume of
liquid injected so as not to dilute the solution too much; [0132]4/
carrying out mixing in order to homogeneously distribute the biological
targets on the micro-array of probes 51.

[0133]FIG. 9 illustrates phase 1/. In a first step, starting from the
system emptied (FIG. 9A), the valves of the conduits 58, 59, 60, 61 are
closed, the valves of the feed port 63 and extraction port 62 are open
and the variable-volume reservoir 64 is at a maximum (valve open) (FIG.
9B). The fixed-volume reservoir 66 is closed. The buffer solution is
injected via the feed port 63, thereby creating a pressure difference
between the inlet and the outlet (FIG. 9B). When the buffer solution
reaches the extraction port 62, the process moves onto the next step.

[0134]In a second step, the valves of the conduits 58, 59, 60, 61 are
open, the valves of the feed port 63 and extraction port 62 are open and
the variable-volume reservoir 64 is at a minimum (=valve closed). The
fixed-volume reservoir 66 remains closed. The injection of buffer
solution via the feed port is continued, and the solution passes into the
chamber via the conduits 60 and 61, fills the chamber and then leaves
again via the conduits 58 and 59 to the extraction port (FIG. 9C).

[0135]The fluid loop is completely filled. During this phase, the system
brings the temperature to that which corresponds to the temperature
required for the targets to fix to the probes.

[0136]It is possible to reverse the two steps, and also to carry them out
at the same time.

[0137]FIG. 10 illustrates the phases 2/ and 3/. First of all, the valves
of the feed port 63 and extraction port 62 are closed. All the other
valves are open, the variable-volume reservoir 64 is at a maximum (valve
open) and the fixed-volume reservoir 66 is opened. The biological targets
(carrier liquid containing reactants) are injected into the fixed-volume
reservoir 66. The volume injected fills the reservoir without it
overflowing (FIG. 10A).

[0138]In order to carry out the mixing, it is necessary to empty the
fixed-volume reservoir 66. It is therefore necessary to extract from the
fluid loop the volume of buffer solution corresponding to the volume
injected into the fixed-volume reservoir, without extracting the
biological material. To do this, the extraction is carried out in a
series of pairs of steps comprising at least one pair of phases (FIGS.
10B and 10C) in the case where the volume injected is equal to the
maximum of the variable volume.

[0139]To this effect, in the next step (FIG. 10B), all the valves are
closed, with the exception of the valve of the extraction port 62. The
variable-volume reservoir decreases to the minimum and a volume of
solution equivalent to the maximum volume of the variable volume is
extracted from the loop.

[0140]The valve of the extraction port 62 then becomes closed and the
valves of the conduits 60 and 61 are open. The variable-volume reservoir
64 increases to the maximum. An equivalent volume is extracted from the
fixed-volume reservoir 66 (FIG. 10C).

[0141]FIG. 11 illustrates the mixing phase 4/. The fluid loop is filled
with buffer solution and contains the biological targets. The mixing
phase consists in distributing the biological targets on the microarray
of probes 51, in a chaotic manner. Whatever the flow induced in the
chamber, it will be laminar and there is a risk that the biological
targets will take the same path and encounter the same probes. In order
for the recognition between the targets and the probes to be more
homogeneous over the surface, a chaotic flow is created from the several
laminar flows, which thus multiplies the chances of "match" between the
targets and the probes.

[0142]The mixing is a succession of 4 steps which are repeated
sequentially throughout the duration of the reaction. Each step comprises
2 substeps: [0143]a first substep during which the variable-volume
reservoir decreases to the minimum. One of the four inlets/outlets is
open and the others are closed. A volume equivalent to the maximum volume
of the variable-volume reservoir is injected into the chamber and the
excess is injected into the fixed-volume reservoir 66. The flow therefore
goes from the variable-volume reservoir to the fixed-volume reservoir;
[0144]a second substep during which the variable-volume reservoir
increases to the maximum, an inlet/outlet different than the previously
open inlet/outlet becomes open, whereas the others are closed. For
example, the inlet/outlet which becomes open is that which is opposite
the inlet/outlet previously open. A volume equivalent to the maximum
volume of the variable-volume reservoir is extracted from the chamber and
the same volume is therefore extracted from the fixed-volume reservoir to
the chamber. The flow goes from the fixed-volume reservoir to the
variable-volume reservoir.

[0145]Thus, for example:

[0146]Phase 1: the solution is injected via the inlet 54 (FIG. 11A) and
the solution is extracted via the outlet 56 (FIG. 11B). Phase 2: the
solution is injected via the inlet 55 (FIG. 1C) and the solution is
extracted via the outlet 57 (FIG. 1D). Phase 3: the solution is injected
via the inlet 56 (FIG. 11E) and the solution is extracted via the outlet
54 (FIG. 11F). Phase 4: the solution is injected via the inlet 57 (FIG.
11G) and the solution is extracted via the outlet 55 (FIG. 11H).

[0147]It is of course entirely possible to contemplate carrying out the
injections and extractions randomly, or else extracting the fluid
simultaneously via a pair of inlets/outlets, and then reinjecting it into
the reaction chamber via another pair of inlets/outlets.

EXAMPLES

[0148]A--Materials and Methods

[0149]Biological Material

[0150]The company Sequentia (Evry, France) supplied the HPLC-purified
oligonucleotides. Each probe was synthesized with an amino group
(--CH2)6--NH2 attached to its 5' end.

[0151]A 25-oligomeric control probe for verifying immobilization, labeled
with Cy3®, and two 12-oligomeric probes (allele a and allele b) which
contained a single central point mutation (G in place of A), were used in
this experiment.

[0152]84-oligomeric single-stranded synthetic DNA targets labeled with
Cy3®, with a sequence complementary to the two 12-oligomeric probes,
were used in this experiment. The targets were diluted in a
6×SSC/0.1% SDS hybridization solution.

[0153]Biochip Fabrication

[0154]Microarrays were fabricated on a RosaSlide substrate (RosaTech,
France) using 25 μM of probes diluted in a 10× PBS solution. The
RosaTech 192-point multimicro-projection apparatus was used to deposit
approximately 5 nl of probe solution at each point, without contact or
cross contamination. The surface of the substrate was inactivated using
the RosaBlock solution (RosaTech, France). This step minimizes the
adsorption during the hybridization. The microarrays were washed using a
1% SDS solution for 30 minutes at 80° C., and then rinsed for 30
minutes with hot water at 80° C. This treatment, which ensures the
reproducibility between microarrays, eliminates the oligonucleotide
probes not covalently bound to the substrate.

[0155]285 deposits were arranged uniformly with a gap of 1 mm on the
42×15 mm2 surface of the substrate. The microarray consists of
identical clusters formed by a checkered design of deposits of the two
alleles. Each cluster is separated by the fluorescence-labeled
verification control probe.

[0156]The Hybridization Station According to the Invention

[0157]The TrayMix micromixer is an automatic active mixing and
hybridization station which is compatible with a standard microscope
slide. The reaction chamber has the following dimensions: 18.8
mm×49 mm×50 μm (width×length×height), and is
sealed by means of a circular seal. The automatic fluidic system of
mixing by chaotic advection is shown schematically in FIG. 7.

[0158]The chaotic mixing is created by means of periodic intersecting
flows produced by means of microvalves inside the reaction chamber. The
feed and extraction ports and the inlets/outlets allow various solutions
(hybridization, washing, decontaminating solutions) to be injected into
the chamber and liquids to be extracted, in particular to the waste
container. A computer controls the operation of the device via a user
software interface. The user simply injects the targets directly into the
reaction chamber through the injection orifice. The system creates mixing
by chaotic advection, without any loss of precious biological sample. The
total volume inside the mixing system is 500 μl. The hybridization
temperature is controlled by means of a heating element which can range
between 20 and 80° C.±0.5° C.

[0159]Hybridization

[0160]1--Passive Hybridization (Static)

[0161]1.1--Method Between Slide and Cover Slip (Manual)

[0162]The results obtained according to the conventional method of
hybridization between slide and cover slip are used as a base threshold
for the comparisons. Ideally, a small amount of target (approximately 50
μl) is placed between the surface of the slide carrying the microarray
and the cover slip. The hybridization is carried out in a confined
environment, under controlled relative humidity and temperature.

[0163]1.2--Micromixer without Chaotic Mixing

[0164]The slide carrying the microarray is placed in the micromixer. For
the hybridization, the device mixer features are turned off and a
homogeneous solution of target is introduced into the reaction chamber.

[0165]2--Passive Hybridization (Dynamic)

[0166]Two distinct hybridization protocols were used independently in
order to demonstrate the advantages of the chaotic mixing inside the
reaction chamber:

[0167]2.1--Automatic Hybridization with a Homogeneous Target Solution:
[0168]1. Manual insertion of the microarray into the reaction chamber.
[0169]2. Automatic bringing to temperature and automatic initializing of
the system with a homogeneous target solution. [0170]3. Automatic mixing
in the reaction chamber. [0171]4. Automatic extraction of the reactants
from the reaction chamber. [0172]5. Manual withdrawal of the microarray.
[0173]6. Automatic washing of the chamber with ultrapure water, in
preparation for successive hybridizations.

[0174]2.2--Automatic Hybridization with Injection of the Target Solution
into the Initialized Mixing Loop [0175]1. Manual insertion of the
microarray into the reaction chamber. [0176]2. Automatic bringing to
temperature and automatic initializing of the system with a buffer
solution. [0177]3. Manual injection of the target solution into the
reaction chamber via the injection orifice. [0178]4. Automatic mixing in
the reaction chamber. [0179]5. Automatic extraction of the reactants from
the reaction chamber. [0180]6. Manual withdrawal of the microarray.
[0181]7. Automatic washing of the chamber with ultrapure water, in
preparation for successive hybridizations.

[0182]The comparison of the results obtained according to these two
protocols was used to demonstrate the advantages of the chaotic mixing
according to the invention. All the hybridization experiments were
carried out in two hours, except the kinetics experiments.

[0183]Washing of Microarrays

[0184]All the hybridized arrays were washed for two minutes in a solution
of 5×SSC and 0.1% SDS, followed by washing in a solution of
2×SSC. The microarrays were then dried by centrifugation at 1500 g
for one minute before being scanned.

[0185]Scanning and Analysis of Results

[0186]The microarrays were scanned, with two PMT gains, using GeneTAC®
LS IV (Genomic Solutions Ltd, Cambridgeshire, UK) at a resolution of 10
μm for scanning the immobilized control probes and the hybridized
probes (allele a and allele b) at subsaturation.

[0187]The fluorescence intensity of each point was analyzed by
segmentation using the TARGET software developed by LEOM
(http://leom.ec-lyon.fr/). The average signal intensity of each point was
measured on the microarray. The coefficient of variation (CV) was
calculated on the basis of the measurements carried out. The CV was
determined from the ratio of the standard deviation of the intensity of
the signals to the average intensity of the signal for the same
population. This coefficient makes it possible to compare the homogeneity
of hybridization over the entire surface of the substrate. The signal
intensities of the Cy3®-labeled control probes for the three
microarrays were used to determine the intrinsic heterogeneity of the
microarrays. The overall CV of the substrate was between 0.10 and 0.15.

[0188]B--Results

[0189]Given that the volumes used in the micromixer are 10 times greater
than in the method between slide and cover slip (500 μl compared with
50 μl), two series of experiments were carried out.

[0190]In a first series of experiments, a similar target concentration was
used under two experimental conditions: [0191]1. Static: between slide
and cover slip (1.1) and in the micromixer with the mixing functions
turned off (1.2). [0192]2. Dynamic: micromixer with the mixing functions
operating.

[0193]In the second series of experiments, the same amount of targets was
used, diluted in 50 μl for the method between slide and cover slip,
and in 500 μl for the methods in the micromixer.

[0194]Effect of the Chaotic Mixing on the Hybridization (Identical
Concentrations)

[0195]In order to determine the impact of the active mixing, the
hybridization results obtained with the micromixer with or without
chaotic mixing were compared. The results show the advantages, in terms
of signal intensity and homogeneity, of the dynamic hybridization
compared with the static hybridization.

[0196]The fluorescence results and the CV of the hybridization between
slide and cover slip serve as a reference. The high CV of this technique
demonstrates the nonuniformity of the hybridization response inherent in
the method between slide and cover slip. Even if the initial target
solution is homogeneous, the CV for hybridization in the mixing loop
without chaotic mixing is even greater (0.56).

[0197]The chaotic mixing makes it possible to reduce the CV for
hybridization almost to the value of the CV intrinsic to the microarrays,
irrespective of whether or not the injected solution is homogeneous.

[0198]Tests with an Identical Amount of Target

[0199]The two static and dynamic hybridization experiments were carried
out with 5 pmol of target.

[0200]Even if the target solution used with the micromixer was 10 times
less concentrated than that used with the method between slide and cover
slip, the micromixer made it possible to obtain results that were
superior in terms of intensity and of CV. The signal/noise ratios (SNRs)
for each type of probe are represented in FIG. 12.

[0201]The dynamic hybridization made it possible to increase the
hybridization specificity for the detection of a single nucleotide
polymorphism (SNP) by increasing the SNR for allele a (represented as
black) and by reducing the SNR for allele b (represented as hatched).

[0202]The overall kinetics of the reaction are an important parameter to
be studied since the target concentrations between the two techniques are
very different. FIG. 13 illustrates the overall hybridization kinetics
for the static (black square) and dynamic (circle) hybridizations. The
two curves are similar, which suggests that the hybridization rates are
of the same order of magnitude. That said, at any moment, the signal
intensity and the standard deviation are improved with the dynamic
hybridization compared with the static hybridization.

[0203]With each method, the asymptotic value for hybridization is not
reached. The reduction in the degree of hybridization is less pronounced
with the chaotic mixing, due to the constant supply of target molecules
on the reaction surface, obtained by the active mixing (target/probe
ratio=50).

[0204]The chaotic mixing results in an improvement in the reaction
kinetics and a rapid distribution of the targets over the entire reaction
surface. After 30 minutes, the hybridization signals in the case of the
active mixing are greater than those obtained in the case of the
technique between slide and cover slip.

[0205]It is noted that, in less than 200 minutes of dynamic hybridization,
results similar to those of an overnight hybridization between slide and
cover slip are obtained. This speed, approximately four times greater for
the dynamic hybridization, is a further advantage of the method according
to the invention, in addition to the improvement in the homogeneity and
to the increase in the signal intensity which are obtained with the
dynamic hybridization.

[0206]The principal advantages of the present invention can be summarized
as follows: [0207]Uniform and homogeneous mixing without a dead zone at
the reaction surface of the support to be processed (visualized by
homogenization and mixing of a fluorescent label in the chamber
initialized with a buffer solution). [0208]Improvement in reaction rates,
compared with the static techniques between slide and cover slip,
optimization of the duration and of the number of reaction cycles.
[0209]Highly uniform biological response over the entire zone processed:
the coefficient of variation of the order of magnitude of the intrinsic
uniformity of the support, i.e. the intrinsic "noise" of the support.
[0210]Automation of the entire method, which results in an improvement in
reproducibility. [0211]Improvement in the specificity between two simple
mutations. [0212]Possibility at any moment of adding reactants to the
reaction chamber, via the injection orifice. This is advantageous, for
example, for double labeling systems (ELISA type). [0213]Possibility of
introducing into the fluid loop a heating element (>90° C.) for
performing denaturations of double-stranded targets or alternatively
breaking the secondary structures of targets outside the reaction
chamber.